Genetic markers for improved meat characteristics in animals (MC4R)

ABSTRACT

Genetic markers in the porcine melanocortin-4 receptor (MC4R) gene are disclosed which are associated with favorable meat quality traits including, drip loss, marbling, pH and color. Further, novel sequence data from regions of the gene are disclosed which may be used in a PCR test to screen for the presence of the marker. The genetic marker may be used to screen animals for breeding purposes which have the desired traits. Kits which take advantage of the PCR test are also disclosed.

GRANT REFERENCE CLAUSE

[0001] This invention was supported at least in part by grants from theUnited States Department of Agriculture through the Iowa Agriculture andHome Economics Experiment Station (IAHEES) and Project Number IOW03148(Hatch Funds). The United States government may have certain rights inthis invention.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of geneticallyevaluating animals by assaying for the presence of at least one geneticmarker which is indicative of one or more traits associated with meatquality. In particular, the method analyzes for variation in themelanocortin-4 receptor (MC4R) gene or other variations associatedtherewith which are indicative of these favorable traits.

BACKGROUND OF THE INVENTION

[0003] Genetic differences exist among individual animals as well asamong breeds which can be exploited by breeding techniques to achieveanimals with desirable characteristics. For example, Chinese breeds areknown for reaching puberty at an early age and for their large littersize, while American breeds are known for their greater growth rates andleanness. Often, however, heritability for desired traits is low, andstandard breeding methods which select individuals based upon phenotypicvariations do not take fully into account genetic variability or complexgene interactions which exist.

[0004] Restriction fragment length polymorphism (RFLP) analysis has beenused by several groups to study pig DNA. Jung et al., Theor. Appl.Genet., 77:271-274 (1989), incorporated herein by reference, disclosesthe use of RFLP techniques to show genetic variability between two pigbreeds. Polymorphism was demonstrated for swine leukocyte antigen (SLA)Class I genes in these breeds. Hoganson et al., Abstract for AnnualMeeting of Midwestern Section of the American Society of Animal Science,Mar. 26-28, 1990, incorporated herein by reference, reports on thepolymorphism of swine major histocompatibility complex (MHC) genes forChinese pigs, also demonstrated by RFLP analysis. Jung et al., Theor.Appl. Genet., 77:271-274 (1989), incorporated herein by reference,reports on RFLP analysis of SLA Class I genes in certain boars. Theauthors state that the results suggest that there may be an associationbetween swine SLA/MHC Class I genes and production and performancetraits. They further state that the use of SLA Class I restrictionfragments, as genetic markers, may have potential in the future forimproving pig growth performance.

[0005] The ability to follow a specific favorable genetic alleleinvolves a novel and lengthy process of the identification of a DNAmolecular marker for a major effect gene. The marker may be linked to asingle gene with a major effect or linked to a number of genes withadditive effects. DNA markers have several advantages; segregation iseasy to measure and is unambiguous, and DNA markers are co-dominant,i.e., heterozygous and homozygous animals can be distinctivelyidentified. Once a marker system is established selection decisionscould be made very easily, since DNA markers can be assayed any timeafter a tissue or blood sample can be collected from the individualinfant animal, or even an embryo.

[0006] The use of genetic differences in receptor genes has become avaluable marker system for selection. For example, U.S. Pat. Nos.5,550,024 and 5,374,526 issued to Rothschild et al. disclose apolymorphism in the pig estrogen receptor gene which is associated withlarger litter size, the disclosure of which is incorporated herein byreference.

[0007] U.S. Pat. No. 5,935,784 discloses polymorphic markers in the pigprolactin receptor gene which are associated with larger litter size andoverall reproductive efficiency. Perhaps one of the most importantcharacteristics for any meat producing animal is meat quality. Meatquality is a difficult characteristic to assess, as many differentaspects, both objective and subjective, make up the overall trait. Thelist of factors which determine quality in meat, as with other foods, israther long (Wood et al., Proceedings of The Nutrition Society (1999)58:363-70). It includes freedom from microbiological hazards (foodsafety) and prevention of animal exploitation (animal welfare). It alsoincludes the sensory appeal of meat, i.e. its taste or eating quality,and perceived healthiness, especially in relation to the amount and typeof fat.

[0008] The quality of raw pig meat is influenced by a large number ofgenetic and non-genetic factors. The latter include farm, transport,slaughter and processing conditions. Meat scientists have performed asubstantial amount of research on these factors, which has led toconsiderable quality improvement. Part of the research has also beendedicated to the genetic background of the pigs, and several studieshave revealed the importance of genetic factors. This has made theindustry aware that selective breeding of pigs and the use of genetechnology can play an important role in enhancing pork quality.

[0009] Information at DNA level can help to fix a specific major gene,but it can also assist the selection of quantitative trait for which wealready select. Molecular information in addition to phenotypic data canincrease the accuracy of selection and therefore the selection response.The size of the extra response in such a Marker Assisted Selection (MAS)program has been considered by many workers from a theoretical point ofview. In general terms, MAS is more beneficial for traits with a lowheritability and which are expensive to measure phenotypically. Meatquality in particular qualifies as an excellent opportunity to utilizeMAS. For example, Meuwissen, T. H. E. and Goddard, M. E.(1996) “The useof Marker Haplotypes in Animal Breeding Schemes”, Genet. Sel. Evol., 28161-176 considered the impact of Marker Assisted Selection for traitssuch as reproduction and meat quality that are difficult to progressusing traditional methods. their results are extremely encouraging,showing that for traits such as meat quality, where the trait ismeasured after slaughter, an additional response of up to 64% could beachieved.

[0010] Indeed, the best approach to genetically improve meat quality isto find relevant DNA-markers directly in the population under selection.Meat quality measurements can be performed continuously on some animalsfrom the nucleus populations of breeding organizations. Since a fullassessment of meat quality can only be done after slaughter, the datamust be collected on culled animals and cannot be obtained on potentialbreeding animals.

[0011] This phenotypic meat quality data is collected in order to enablethe detection of relevant DNA markers, and to validate markers fromexperimental populations or to test candidate genes. Significant markersor genes can then be included directly in the selection process. Anadvantage of the molecular information is that we can obtain it alreadyat very young age of the breeding animal, which means that animals canbe preselected based on DNA markers before the growing performance testis completed. This is a great advantage for the overall testing andselection system.

[0012] It can be seen from the foregoing that a need exists for a methodfor improving meat quality characteristics in animals by identifying andselecting animals with the improved meat characteristics.

[0013] An object of the present invention is to provide a genetic markerbased on or within the MC4R gene which is indicative of favorable meatcharacteristics such as those evidenced by pH, marbling, color and driploss.

[0014] Another object of the invention is to provide an assay fordetermining the presence of this genetic marker.

[0015] A further object of the invention is to provide a method ofevaluating animals that increases accuracy of selection and breedingmethods for the desired traits.

[0016] Yet another object of the invention is to provide a PCRamplification test which will greatly expedite the determination ofpresence of the marker.

[0017] An additional object of the invention is to provide a kit forevaluating a sample of animal DNA for the identified genetic marker.

[0018] These and other objects, features, and advantages will becomeapparent after review of the following description and claims of theinvention which follow.

SUMMARY OF THE INVENTION

[0019] This invention relates to the discovery of a polymorphism withinthe melanocortin-4 receptor (MC4R) gene which is associated with meatquality traits in animals. This gene is highly conserved among speciesand it is expected that the different alleles disclosed herein will alsocorrelate with variability in this gene in other meat producing animalssuch as bovine, sheep, chicken, etc. This polymorphic site has beenpreviously described in an earlier patent application PCT/US99/16862,publication number WO 00/06777 the disclosure of which is incorporatedherein. In the earlier application this site was found to significantlycorrelate with weight gain and feed intake, in other words, traitsinvolving growth rate of the pig. Surprisingly, as fast growth isgenerally considered to be negatively correlated with meat quality, themarker has now been shown to correlate with favorable meatcharacteristics such as pH level, marbling, color, and drip loss. Thesemultigenic characteristics have been previously difficult to associatewith quantitative trait loci and current improvements in meatcharacteristics have centered around understanding and controlling thenumerous factors i.e. on a farm, transport, and/or slaughter planthandling influencing meat quality including the incidence of PSE (pale,soft, exudative), RSE (red, soft, exudative), and DFD (dark, firm, dry)meat. According to the invention, the association of the MC4Rpolymorphism with the these trait(s) enables genetic markers to beidentified for specific breeds or genetic lines to identify animals withfavorable meat characteristics early in the animal's life.

[0020] The marker genotype consists of a polymorphism within the MC4Rgene that results in a guanine to adenine transition and a missensemutation of aspartic acid(D) codon (GAU) into asparagine(N) codon (AAU)at a position corresponding to amino acid position 298 of the human MC4Rprotein resulting in a TaqI restriction site in one allele of the gene.In one embodiment of the invention a TaqI restriction pattern whichidentifies the polymorphism is used to assay for the presence or absenceof markers associated with the desirable meat traits. The inventionincludes assays for detection of the marker, or markers linked theretoas well as the sequence characterization of the polymorphism andincludes novel sequences in the MC4R gene which may be used to designamplification primers for such an assay (SEQ ID NO:1). Additionally, theinvention includes a method for using the assay in breeding programs foranimal selection and a kit for performing the assay.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is the sequence listing for MC4R in pigs (SEQ ID NO:1). “X”represents the site of the polymorphism.

[0022]FIG. 2 represents a comparison of the DNA sequence between thehuman (SEQ ID NO:2) and the porcine (SEQ ID NO:3) MC4R gene.

[0023]FIG. 3 represents a comparison of the amino acid sequence betweenthe human (SEQ ID NO:4) and the porcine (SEQ ID NO:5) MC4R gene.

[0024]FIGS. 4a, 4 b, and 4 c are linkage reports for MC4R from CRI-MAP.

[0025]FIG. 5 depicts partial nucleotide and amino acid sequences (SEQ IDNO:12) of the porcine MC4R gene. The amino acid translation shows anamino acid substitution at codon 298.

[0026]FIG. 6 depicts multiple-alignments of the putative seventhtransmembrane domain of porcine MC4R with other MCRs and GPCRs. The “*”represents the predicted sequence positions for porcine MC4R. The otheramino acid sequences were obtained from the GenBank database (accessionnumbers P32245, P70596, P41983, P56451, P34974, P41968, P33033, Q01718,Q01726, Q28031, AF011466, P21554, P18089, P30680, P47211). The missensevariant in porcine MC4R substituted amino acid N for D in the positionmarked with an arrow. The Asp (D) residue is highly conserved amongMCRs, and the Asn (N) residue is well conserved in most other GPCRs.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The melanocortin-4 receptor (MC4R) has been shown to be animportant mediator of long term weight homeostasis. MC4R antagonists canincrease food intake and body weight during chronic administration.Skuladottir, G. V., et al., “Long term orexigenic effect of a novelmelanocortin 4 receptor selective antagonist”, British J. of Pharm.,126(1):27-34 (1999).

[0028] Lu et al., Nature (Oct. 27, 1994), 371 (6500):799-802 suggestedthat the melanocortin receptors are involved in controlling food intakeand energy balance through studying its antagonism to the agouti obesitysyndrome. Huszar et al., Cell (Jan. 10, 1997) 88(1):131-41 found thatinactivation of the melanocortin-4 receptor gene (MC4R) resulted in amaturity onset obesity syndrome in mice and demonstrated a major role ofMC4R protein in the regulation of energy balance related to the agoutiobesity syndrome. In addition, the MC4R protein mediates the effects ofleptin, one of the important signaling molecules in energy homeostasis(Seeley et al. 1997).

[0029] According to the present invention, a variant or polymorphism inthe MC4R gene has been located, and this genetic variability isassociated with phenotypic differences in the porcine meat qualitytraits as evidenced by pH, marbling, color and drip loss.

[0030] In one embodiment of the invention, an assay is provided fordetection of presence of a desirable genotype in animals. The assayinvolves assaying the genomic DNA purified from blood, tissue, semen, orother convenient source of genetic material by the use of primers andstandard techniques, such as the polymerase chain reaction (PCR), thendigesting the DNA with a restriction enzyme (e.g., Taq I or other enzymewhich cleaves at the same G→A site) so as to yield gene fragments ofvarying lengths, and separating at least some of the fragments fromothers (e.g., using electrophoresis).

[0031] The fragments may also be detected by hybridizing with anucleotide probe (e.g., radio-labeled cDNA probes) that contains all orat least a portion of the MC4R gene cDNA sequence to the separatedfragments and comparing the results of the hybridization with assayresults for a gene sequence known to have the marker or a sequence knownto not have the marker. Selection and use of probes for detection ofMC4R sequences based on the known and disclosed MC4R sequences isgenerally known to those skilled in the art. The probe may be anysequence which will hybridize to the separated digestion products andallow for detection.

[0032] Another embodiment of the invention provides a kit for assayingthe presence in a MC4R gene sequence of a genetic marker. The markerbeing indicative of heritable traits of meat quality characteristics.The kit in a preferred embodiment also includes novel PCR primerscomprising 4-30 contiguous bases on either side of the polymorphism toprovide an amplification system allowing for detection of the G→ATransition polymorphism by PCR digestion of PCR products. The sequencesurrounding the polymorphic site is shown in SEQ ID NO:1, FIG. 1.Several primers have also been disclosed including SEQ ID NOS:6 and 7,SEQ ID NOS:10 and 11; and mapping primers 8 and 9. The preferred primersare SEQ ID NO:10 and SEQ ID NO:11.

[0033] A further embodiment comprises a breeding method whereby an assayof the above type is conducted on a plurality of DNA samples fromdifferent animals or animal embryos to be selected from and based on theresults, certain animals are either selected or dropped out of thebreeding program.

[0034] According to the invention, in a preferred embodiment, thepolymorphism in the MC4R gene identifiable by the Taq I restrictionpattern, is disclosed. As is known in the art, restriction patterns arenot exact determinants of the size of fragments and are onlyapproximate. When the primers SEQ ID NOS:6 and 7 are used thepolymorphism is identifiable by three bands from a Taq I digestion ofthe PCR product, 466, 225, and 76 base pairs (bp) for one homozygousgenotype (allele 1); two bands, 542 and 225 bp for another homozygousgenotype (allele 2); and four bands for the heterozygous genotype (542,466, 225, and 76 bp). When the preferred primers are used, SEQ ID NOS:10and 11 the bands upon taq digestion include 156 and 70 bp for allele 1and one 226bp fragment for allele 2. Those of skill in the art willappreciate that the design of alternate primers PCR conditions andrestriction patterns for identifying the presence of allele 2 using theMC4R sequence data herein or other data for closely linked locirepresent nothing more than routine optimization of parameters and areintended to be within the scope of the invention. The marker forimproved meat characteristics as evidenced by all four meat qualitymeasurements observed herein (allele 2). The allele 2 genotype waspreviously associated with faster growth rate. This is surprisingbecause the current state of the art concluded that there is a negativecorrelation between growth rate and meat quality.

[0035] In addition, the polymorphism associated with the pattern hasbeen identified at the nucleotide level. The polymorphic Taq I site wassequenced along with the general surrounding area. See SEQ ID NO: 1. Thesequences surrounding the polymorphism have facilitated the developmentof a PCR test in which a primer of about 4-30 contiguous bases takenfrom the sequence immediately adjacent to the polymorphism is used inconnection with a polymerase chain reaction to greatly amplify theregion before treatment with the Taq I restriction enzyme. The primersneed not be the exact complement; substantially equivalent sequences areacceptable.

[0036] From sequence data, it was observed that in allele 2 a guanine issubstituted with an adenine at position 678 of the PCR product shown inFIG. 1 or at position 298 of the analogous human MC4R amino acid of theMC4R protein changing the aspartic acid codon (GAU) into an asparaginecodon (AAU). The PCR test for the polymorphism used a forward primer of5′-TGG CAA TAG CCA AGA ACA AG-3′ (SEQ. ID NO: 6) and a reverse primer of5′-CAG GGG ATA GCA ACA GAT GA-3′ (SEQ. ID NO: 7). Pig specific primersused for physical mapping were a forward primer of 5′-TTA AGT GGA GGAAGA AGG-3′ (SEQ. ID NO: 8) and a reverse primer of 5′-CAT TAT GAC AGTTAA GCG G-3′ (SEQ ID NO:9). The resulting amplified product of about 750bp, when digested with Taq I, results in allelic fragments of 466, 225,and 76 bp (allele 1) or 542 and 225 bp (allele 2). The most preferredprimers resulting in either 2 or 1 fragment after Taq I digestion areSEQ ID NOS:10 and 11. Allele 1 generates fragments of 156 and 70 basepairs while allele 2 generates a single 226 bp fragment.

[0037] The marker may be identified by any method known to one ofordinary skill in the art which identifies the presence or absence ofthe particular allele or marker, including for example, single-strandconformation polymorphism analysis (SSCP), RFLP analysis, heteroduplexanalysis, denaturing gradient gel electrophoresis, allelic PCR,temperature gradient electrophoresis, ligase chain reaction, directsequencing, minisequencing, nucleic acid hybridization, andmicro-array-type detection of the MC4R gene and examination for thepolymorphic site. Yet another technique includes an Invader Assay whichincludes isothermic amplification that relies on a catalytic release offluorescence. See Third Wave Technology at www.twt.com all of which areintended to be within the scope of the invention.

[0038] One or more additional restriction enzymes and/or probes and/orprimers can be used. Additional enzymes, constructed probes, and primerscan be determined by routine experimentation by those of ordinary skillin the art and are intended to be within the scope of the invention.

[0039] Other possible techniques include non-gel systems such as TaqMan™(Perkin Elmer). In this system, oligonucleotide PCR primers are designedthat flank the mutation in question and allow PCR amplification of theregion. A third oligonucleotide probe is then designed to hybridize tothe region containing the base subject to change between differentalleles of the gene. This probe is labeled with fluorescent dyes at boththe 5′ and 3′ ends. These dyes are chosen such that while in thisproximity to each other the fluorescence of one of them is quenched bythe other and cannot be detected. Extension by Taq DNA polymerase fromthe PCR primer positioned 5′ on the template relative to the probe leadsto the cleavage of the dye attached to the 5′ end of the annealed probethrough the 5′ nuclease activity of the Taq DNA polymerase. This removesthe quenching effect allowing detection of the fluorescence from the dyeat the 3′ end of the probe. The discrimination between different DNAsequences arises through the fact that if the hybridization of the probeto the template molecule is not complete, i.e., there is a mismatch ofsome form, the cleavage of the dye does not take place. Thus, only ifthe nucleotide sequence of the oligonucleotide probe is completelycomplementary to the template molecule to which it is bound willquenching be removed. A reaction mix can contain two different probesequences each designed against different alleles that might be present,thus, allowing the detection of both alleles in one reaction.

[0040] Though the use of RFLPs is one method of detecting thepolymorphism, other methods known to one of ordinary skill in the artmay be used. Such methods include ones that analyze the polymorphic geneproduct and detect polymorphisms by detecting the resulting differencesin the gene product.

[0041] Though the preferred method of separating restriction fragmentsis gel electrophoresis, other alternative methods known to one skilledin the art may be used to separate and determine the size of therestriction fragments.

[0042] It is possible to indirectly select for the polymorphism withalternative DNA markers and these methods are also within the scope ofthe invention. One can establish a linkage between specific alleles ofalternative DNA markers and alleles of DNA markers known to beassociated with the MC4R gene which have previously been shown to beassociated with a particular trait. Examples of markers on the publishedPiGMaP chromosome map which are linked to the MC4R gene include S0331,BHT0433, and S0313. This is also true for other species as well, forexample in human the MC4R gene is located at chromosome 18q21.3-q22.

[0043] The reagents suitable for applying the methods of the presentinvention may be packaged into convenient kits. The kits provide thenecessary materials, packaged into suitable containers. At a minimum,the kit contains a reagent that identifies the polymorphism in the MC4Rgene that is associated with the trait of meat quality. Preferably, thereagent that identifies the polymorphism is a PCR set (a set of primers,DNA polymerase, and four nucleoside triphosphates) that hybridize withthe MC4R gene or a fragment thereof. Preferably, the PCR set andrestriction enzyme that cleaves the MC4R sequence in at least one placeare included in the kit. Preferably, the kit further comprisesadditional means, such as buffers or reagents, for detecting ormeasuring the detectable entity or providing a control. Other reagentsused for hybridization, prehybridization, DNA extraction, visualization,and similar purposes may also be included, if desired.

[0044] The genetic markers, methods, and kits of the invention areuseful in a breeding program to identify and/or to select for meatcharacteristics in a breed, line, or population of animals. Continuousselection and breeding of animals that are at least heterozygous andpreferably homozygous for the desired polymorphism associated with theparticular trait would lead to a breed, line, or population having thosedesired traits. Thus, the marker is a selection tool.

[0045] The following examples are offered to illustrate, but not limitthe invention.

EXAMPLE 1 Melanocortin 4 Receptor PCR-RFLP Test—TaqI Polymorphism andGenetic Linkage Mapping of MC4R Gene

[0046] Primers:

[0047] Primers were designed from homologous regions of human and ratMC4R sequences (Genbank Accession No. s77415 and u67863, respectively).These primers were used to amplify a 750-bp sequence of the porcine MC4Rgene. MC4R1: 5′ TGG CAA TAG CCA AGA ACA AG 3′ (SEQ ID NO:6) MC4R4: 5′CAG GGG ATA GCA ACA GAT GA 3′ (SEQ ID NO:7)

[0048] PCR Conditions: Mix 1: 10 × Promega Buffer 1.0 μL 25 mM MgCl₂ 0.6μL dNTPs mix (2.5 mM each) 0.5 μL 25 pmol/μL MC4R1 0.1 μL 25 pmol/μLMC4R4 0.1 μL dd sterile H₂O 7.5 μL Taq Polymerase (5 U/μL) 0.07 μL Genomic DNA (12.5 ng/μL) 1.0 μL

[0049] Ten μL of Mix 1 and DNA were combined in reaction tube, thenoverlaid with mineral oil. The following PCR program was run: 94° C. for2 min.; 35 cycles of 94° C. for 30 sec.; 58° C. 1 min., and 72° C. 1min. 30 sec.; followed by a final extension at 72° C. for 15 min.

[0050] Five μl of the PCR reaction product was checked on a standard 1%agarose gel to confirm amplification success and clean negative control.Product size is approximately 750 base pairs. Digestion was performed bythe following procedure. TaqI Digestion Reaction 10 μL reaction PCRproduct 5.0 μL 10 × TaqI NE Buffer 1.0 μL BSA (10 mg/ml) 0.1 μL TaqIenzyme (20 U/μL) 0.5 μL dd sterile H₂O 3.4 μL

[0051] A cocktail of the buffer, enzyme, BSA, and water was made. FiveμL was added to each reaction tube containing the DNA. The mixture wasthen incubated at 65° C. for at least 4 hours to overnight. Loading dyewas mixed with the digestion reaction and the total volume was loaded ona 3% agarose gel. The major bands for allele 1 are about 466, 225, and76 bp. The allele 2 genotype bands are 542 and 225 bp. The heterozygotegenotype has both allele 1 and allele 2.

[0052] Results

[0053] The amplified PCR product is about 750 bp. The sequence of thePCR product confirmed that the PCR product is MC4R gene with 97.6%, and92.2% identities at the amino acid and DNA level, respectively, tocorresponding human sequences. (see FIGS. 2 and 3).

[0054] The TaqI digestion of the PCR product produced allelic fragmentsof 466, 225, and 76 bp (allele 1), or 542 and 225 bp (allele 2). Theheterozygote genotype has both types of alleles. Mendelian inheritancewas observed in three three-generation international reference families,which were used to map this gene by linkage analysis.

[0055] The polymorphism between allele 1 and allele 2 resulting from aG→A transition at position 678 of the PCR product revealed a missensemutation of Aspartic acid codon (GAU) into Asparagine codon (AAU) atposition 298 amino acid of MC4R protein. (See FIG. 1, SEQ ID NO:1).

[0056] Allele frequencies were determined by genotyping of DNA samplesfrom a small number of animals from different breeds (Table 1). Allele 1was observed with a frequency of 1 in Meishan, but was not observed orobserved at very low frequency in Hampshire and Yorkshire. Thefrequencies of allele 1 in Landrace and Chester White were 0.5,respectively.

[0057]FIGS. 2 and 3 illustrate the differences between the DNA and aminoacid sequences of the human and porcine MC4R gene (SEQ ID NOS:2-5).TABLE 1 The Frequency of Allele 1 in Different Pig Breeds Breed #Animals Freq. Allele 1 Meishan 8 1 Large White 8 0.56 Yorkshire 6 0.08Hampshire 5 0 Landrace 4 0.5 Chester White 4 0.5 Minzu 2 1 Wild Boar 2 1

[0058] Linkage Analyses

[0059] Two-point and multi-point linkage analyses were performed on thegenotypes of international reference families. See FIGS. 4a-4 c. Thedata were analyzed by using the CRI-MAP program. MC4R was significantlylinked to several markers on porcine chromosome (SSC) 1. The mostclosely linked markers (recombination fraction and LOD score inparentheses) are S0331 (0.02, 21.97), BHT0433 (0.02, 21.32), and S0313(0.00, 17.76) by two-point linkage analysis. A multi-point linkageanalysis produced the best map order of markers and MC4R (with distancein Kosambi cM): KGF-5.8-CAPN3-2.5-MEF2A-6.1-MC4R-5.6-S0313.

[0060] Somatic cell hybrid panel of pig and rodent was used to assignMC4R to a cytogenetic region. PCR products from pig specific primerswere amplified in clones 7, 8, 16, 18, and 19. MC4R was localized toSSClq 22-27.

EXAMPLE 2 Discovery of a Missense Variant of the Porcine Melanocortin-4Receptor (MC4R) Gene

[0061] To determine if there was an association of this MC4Rpolymorphism with phenotypic variation the mutation was tested in alarge number of individual animals from several different pig lines.Analyses of growth and performance test records showed significantassociations of MC4R genotypes with backfat, growth rate and feed intakein a number of lines. It is probable that the variant amino acid residueof the MC4R mutation causes a significant change of the MC4R function.These results support the functional significance of a pig MC4R missensemutation and suggest that comparative genomics based on model speciesmay be equally important for application to farm animals as they are forhuman medicine.

[0062] Identification of mutations in the leptin and the leptin receptorhas provided some information on genetic components involved in theregulation of energy balance (Zhang et al. 1994; Tartaglia et al. 1995).Genetic studies using animal models have facilitated the identificationof major genetic causes of obesity (Andersson 1996; Pomp 1997;Giridharan 1998). Furthermore, several other genes involved in theneural signaling pathway of energy homeostasis have been identified(Flier and Maratos-Flier 1998; Schwartz et al. 1999). Of particularinterest among candidate signaling molecules involved in the regulationof energy homeostasis is the melanocortin-4 receptor (MC4R). The MC4Rresponse to leptin signaling is a link between food intake and bodyweight (Seeley et al. 1997; Marsh et al. 1999). Neuropeptide Y (NPY)signaling in the central nervous system is also mediated by the MC4Rprotein (Kask et al. 1998). Several mutations in MC4R includingframeshift and nonsense mutations are associated with dominantlyinherited obesity in humans (Vaisse et al. 1998; Yeo et al. 1998). Someother MC4R missense mutations in humans have also been identified(Gotoda et al. 1997; Hinney et al. 1999) but the functional significanceof these mutations has not been characterized.

[0063] Selection based on growth characteristics has been of greatimportance to the pig industry because of costs associated with feedingand consumer preference for lean meat. Efficient genetic improvement inthese quantitative traits may be augmented through the use of markerassisted selection (MAS) using high density genetic maps (Dekkers andvan Arendonk 1998; Rothschild and Plastow 1999). An important tool inthis process is comparative mapping using the well-developed human andmouse gene maps, which assist in the identification of correspondinggenomic regions or major genes controlling growth and performance traitsin the pig. Biological understanding of complex traits in human or modelspecies offers an alternative approach to identify genes responsible forthe traits of economic interest in livestock. Several quantitative traitloci (QTL) linkage scans using phenotypically divergent breeds andcandidate gene analyses have been successfully conducted for fatness andgrowth traits (Yu et al. 1995; Casas-Carrillo et al. 1997; Knorr et al.1997; Knott et al. 1998; Rohrer et al. 1998; Wang et al. 1998; Paszek etal. 1999), but no individual genes with major effects on growth andperformance traits have yet been established for commercial populations.The role of MC4R in feed intake and obesity suggests it may be animportant genetic marker for the growth-related traits in the pig.

[0064] Materials and Methods

[0065] Animals. Pigs were raised under normal production conditionsunder the care of PIC employees in nucleus farms in the United Statesand Europe.

[0066] PCR amplification of a pig MC4R gene fragment. Primers weredesigned from homologous regions of human and rat MC4R sequences(GenBank accession no. s77415 and u67863, respectively). The primerswere: forward primer: 5′-TGG CAA TAG CCA AGA ACA AG-3′ (SEQ. ID NO:6)and reverse primer: 5′-CAG GGG ATA GCA ACA GAT GA-3′ (SEQ. ID NO:7). ThePCR reaction was performed using 12.5 ng of porcine genomic DNA, 1×PCRbuffer, 1.5 mM MgCl₂, 0.125 mM dNTPs, 0.3 mM of each primer, and 0.35 UTaq DNA polymerase (Promega) in a 10 μL final volume. The conditions forPCR were as follows: 2 min at 94° C.; 35 cycles of 30 s at 94° C., 1 minat 56° C., 1 min 30 s at 92° C., and a final 15 min extension at 72° C.in a Robocycler (Stratagene, La Jolla, Calif.).

[0067] Sequencing and mutation detection. Sequencing of the PCR productsfrom several individual pigs of different breeds was conducted and thesequences were compared to detect any nucleotide change. Sequencing wasperformed on an ABI sequencer 377 (Applied Biosystems). The porcine MC4Rsequence has been submitted to GenBank, and has accession numberAF087937. The sequence analysis revealed one nucleotide substitutionsituated within a TaqI restriction enzyme recognition site (Kim et al.1999). A set of primers was then designed to generate a smaller MC4Rgene fragment, which contained only one informative TaqI restrictionsite to specify the polymorphic site and to facilitate the PCR-RFLPtest. These primers were: forward 5′-TAC CCT GAC CAT CTT GAT TG-3′ (SEQ.ID NO:10) and reverse: 5′-ATA GCA ACA GAT GAT CTC TTT G-3′ (SEQ. IDNO:11).

[0068] Results

[0069] Identification of a missense mutation in the pig MC4R gene. TheMC4R gene consists of approximately 1 kb of coding sequence containedwithin a single exon. About 750 bp of a pig MC4R gene fragment wasproduced by PCR (Kim et al. 1999). The sequence of the PCR productconfirmed that the PCR product is the MC4R gene with 92.2% and 97.6%identities at nucleotide and the amino acid levels, respectively, to thehuman MC4R sequence. Multiple alignments of the sequences fromindividual animals of several breeds identified a single nucleotidesubstitution (G→A; FIG. 5). The polymorphism revealed a missensemutation that replaces aspartic acid (GAU) with asparagine (AAU) at theposition identical to amino acid 298 of human MC4R protein. To confirmthis base change, we designed pig-specific primers flanking thepolymorphic site and analyzed the polymorphism as a TaqI PCR-RFLP gel(FIG. 6). FIG. 6 shows a TaqI digestion of the PCR product analyzed byagarose-gel electrophoresis. Allele 1 produced 156 and 70 bp fragmentsand allele 2 produced a 226 bp fragment as the PCR-RFLP. Theheterozygote has both allele 1 and 2 fragments. Molecular marker (M) andMC4R genotypes are indicated at the top of each lane.

[0070] The MC4R missense mutation is within a highly conserved regionamong melanocortin receptors (MCR). The MCR is a subfamily of G-proteincoupled receptors (GPCR) containing certain conserved structuralelements common to most other GPCRs, but overall amino acid identitiesbetween MCR and other GPCRs are low (Tatro 1996). A multiple-alignmentof the predicted amino acid sequences of the pig MC4R with MC4R proteinsfrom other species, other MCR proteins, or representative GPCRs showedthat the aspartic acid found at position 298 of the seventhtransmembrane domain is very highly conserved in the MCR proteins (FIG.7). It is interesting to note, however, that this position is occupiedby asparagine in most other GPCRs. The MCR proteins show 40-80% aminoacid identity with each other (Tatro 1996), but the secondintracytoplasmic loop and the seventh transmembrane domain are highlyconserved among MCR proteins (Gantz et al. 1993). Some of therelationships between MCR structure and function have been discovered bythe studies of natural and experimental mutations in humans and mice(Robbins et al. 1993; Valverde et al. 1995; Frandberg et al. 1998).These studies indicate that some mutations in highly conserved regionscause structural changes and alter the function of the receptor. TheAsp298Asn substitution mutation could have an effect on the function ofthe receptor. However, this will require further testing but it is knownthat change of the homologous residue in MClR (Asp294His) is associatedwith fair skin and red hair in humans (Valverde et al. 1995).

EXAMPLE 3

[0071] Quantity and quality are descriptive terms of great importance inthe meat industry. As the live animal is converted to meat and the meatmoves along the line of distribution, from slaughters and processors toretailers and finally to consumers, the factor of quality becomesincreasingly more important. Obviously, economic considerationsinfluence the concerns for quantity and quality.

[0072] The condition of pale, soft and exudative (PSE) pork andgenerally very high variability of pork quality was recognized anddocumented by 1960, and both quality “defects” have been viewed ashaving less value for further processing and being inferior forconsumers. Although an enormous amount of research has been directed atthe problem through a half-century of effort, surveys of incidenceshowed, in pork produced in the U.S.A., that 18% was of inferior quality(PSE) in 1963 and 16% in 1992. Thus, the existence of gene markersassociated with both the ability to change the levels of traits (i.g.meat color, water holding capacity, tenderness or marbling) as well asto reduce variation in meat quality characteristics provides excellentopportunities for a dramatic improvement in meat quality. First, genemarkers allow for significant steps to be made in the desired directionof quality traits; (e.g. improving technological yield of processed hamand reducing moisture (purge) losses of fresh ham and loins, byselecting against the RN gene in pigs). Secondly, gene markers will helpreduce meat quality variation, since we can fix relevant genes in thebreeding populations.

[0073] Meat quality is typically measured in slaughter plants in termsof the pH of the meat, color (using several different instruments andmethods e.g. Minolta, [Min]), marbling and drip loss.

[0074] For example, the following descriptions of desirable meat qualitycharacteristics are generally accepted by the industry based on theireconomic value at different segments of the pork supply chain:

[0075] Loin Minolta Lightness (L*): The range of 43-47 units (fromdarker to lighter color) is acceptable, but L* of 43 is better; i.e.,has higher economic value, in general in this range**

[0076] Loin Japanese Color Score (JCS): The range of 2.5-5.0 units (fromlighter to darker color) is acceptable, but JCS of 3-4 is better

[0077] Loin Marbling (level of intramuscular fat): Generally, highermarbling is better as it is associated with improved meat eating qualitycharacteristics

[0078] Loin pHu: (ultimate meat acidity measured 24 hours post-mortem;this attribute is the single most important trait of pork quality);--Therange of 5.50-5-80 is desirable, but 5.80 is better as it positivelyinfluences the color and (low) purge of the meat

[0079] Ham Minolta lightness (L*) The range of 43-52 units isacceptable, but lower (43) is better

[0080] Ham pHu: higher; i.e., 5.80, is better

[0081] Drip loss or purge: the range of 1%-3% is acceptable, but loweris better ** this may be dependent upon market, for example in Japandarker pork is preferred. Sosnicki, A. A., E. R. Wilson, E. B. Sheiss,A. devries, 1998 “Is there a cost effective way to produce high qualitypork?”, Reciprocal Meat Conference Proceedings, Vol. 51.

[0082] Results TABLE 1 Least square means for different MC4R genotypicclasses based on a sample of 1146 animals from six genetic lines(preferred class in bold) Genotype Trait 11 12 22 p value Loin pHu 5.705.70  5.73 <0.01 Ham pHu 5.69 5.69  5.72 <0.07 Ham Min L 48.44 48.3947.38 <0.03 Drip 2.29 2.43  2.10 <0.07 Loin Marbling 2.17 2.18  2.25<0.42 Days to 110 g* 169.2 168.5 166.4  <0.0001

[0083] Significant effects of marker genotype are identified forultimate pH (pHu), color (Min) and drip loss and a desirable trend isobserved for marbling. The size of the effects observed betweengenotypes while small are of commercial significance in terms ofdifferences in meat quality. It can be seen from the results in Table 1that allele 2 is the preferred allele in this sample for all four meatquality measures. Interestingly, this is the preferred allele for growthas reported in WO 00/06777. This is a particularly important finding, asit is somewhat unexpected. In general, there is a negative correlationbetween growth rate and meat quality. Indeed, there is a generalperception that meat quality has decreased as breeders have selected forincreased growth rate.

[0084] In some situations we might anticipate that the associationsbetween the marker genotype and the traits may differ in direction. Thiswill be the case where the marker utilized here is linked to thepolymorphism or gene that is causing the effect. In this situation, theMC4R marker will still have utility, once the association has beenidentified by experimentation.

[0085] The traits measured here are only some of the measures that maybe used for determining meat quality. Many others can be used that arecorrelated to these measures. Thus it will be expected that similareffects will be observed for such economically important traits as waterholding and binding capacity, curing and cooking yields and that thesetraits and that these will also extend to related measures of eatingquality such as tenderness, juiciness, flavor and taste. See Sosnicki,supra.

[0086] The present invention concerns the identification of significantassociations between the MC4R marker genotype and meat quality. It willbe realized by those skilled in the art that other gene markers locatedin this region of the swine genome (swine chromosome 1) will also besuitable for marker assisted selection of these traits.

EXAMPLE 4

[0087] A total of 257 animals from a Pietrain-based line of pigs wereslaughtered and meat quality characteristics determined at the time ofslaughter and during post-slaughter handling/conditioning for meatproduction. MC4R genotypes were determined using methods disclosedherein. Associations between marker genotype and MQ traits were thencalculated. The results are depicted in Table 2.

[0088] Significant associations were obtained for ultimate pH and color(Minolta L) of the ham. Hams from animals of genotype 2,2 will bepreferred in markets which favor these characteristics.

[0089] The association can be used to select the parents of slaughterpigs or to improve breeding stock by within line selection.Alternatively, ham processors may chose to purchase pigs of thepreferred genotype in order to improve the overall quality of theproduct (2,2 carcasses will be a better color and will be expected toprovide greater yield than those from 1,1 or 1,2 animals). In addition,by selecting a single genotype they will also reduce the variation inproduct quality due to the different MC4R genotypes. TABLE 2 MC4Rgenotype and Ham pHu and Min L for a Pietrain based line selected to befree of the Halothane gene Genotype n pHu Min L 11 119 5.72 49.70 12 1015.73 50.03 22  37 5.80 47.83 p <0.04    <0.09  

[0090] The present invention describes an association between MC4Rgenotype and meat quality characteristics such as pH, color and marblingin the pig. These traits are in turn associated with visual appearanceand processing and eating quality characteristics such as tenderness.

[0091] These traits also describe meat quality in other species such asbeef and lamb. Because of the relatively close evolutionary link betweenpigs and other meat species it can be predicted that variation in thisgene is also likely to be associated with meat quality (MQ) in theseother species. Polymorphisms can be identified in the MC4R gene of thesespecies using the same approach set out here and the resulting SNPs usedfor association analysis.

[0092] Having described the invention with reference to particularcompositions, theories of effectiveness, and the like, it will beapparent to those of skill in the art that it is not intended that theinvention be limited by such illustrative embodiments or mechanisms, andthat modifications can be made without departing from the scope orspirit of the invention, as defined by the appended claims. It isintended that all such obvious modifications and variations be includedwithin the scope of the present invention as defined in the appendedclaims. The claims are meant to cover the claimed components and stepsin any sequence which is effective to meet the objectives thereintended, unless the context specifically indicates to the contrary.

[0093] The following citations are hereby incorporated in their entiretyby reference:

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1 26 1 746 DNA Sus scrofa 1 acaagaatct gcattcaccc atgtactttt tcatctgtagcctggctgtg gctgatatgc 60 tggtgagcgt ttccaatggg tcagaaacca ttgtcatcaccctattaaac agcacggaca 120 cggacgcaca gagtttcaca gtgaatattg ataatgtcattgactcagtg atctgtagct 180 ccttactcgc ctcaatttgc agcctgcttt cgattgcagtggacaggtat tttactatct 240 tttatgctct ccagtaccat aacattatga cagttaagcgggttggaatc atcatcagtt 300 gtatctgggc agtctgcacg gtgtcgggtg ttttgttcatcatttactca gatagcagtg 360 ctgttattat ctgcctcata accgtgttct tcaccatgctggctctcatg gcttctctct 420 atgtccacat gttcctcatg gccagactcc acattaagaggatcgccgtc ctcccaggca 480 ctggcaccat ccgccaaggt gccaacatga agggggcaattaccctgacc atcttgattg 540 gggtctttgt ggtctgctgg gcccccttct tcctccacttaatattctat atctcctgcc 600 cccagaatcc atactgtgtg tgcttcatgt ctcactttaatttgtatctc atcctgatca 660 tgtgtaattc catcatcrat cccctgattt atgcactccggagccaagaa ctgaggaaaa 720 ccttcaaaga gatcatctgt tgctat 746 2 840 DNAHomo sapiens 2 atatcttagt gattgtggca atagccaaga acaagaatct gcattcacccatgtactttt 60 tcatctgcag cttggctgtg gctgatatgc tggtgagcgt ttcaaatggatcagaaacca 120 ttatcatcac cctattaaac agtacagata cggatgcaca gagtttcacagtgaatattg 180 ataatgtcat tgactcggtg atctgtagct ccttgcttgc atccatttgcagcctgcttt 240 caattgcagt ggacaggtac tttactatct tctatgctct ccagtaccataacattatga 300 cagttaagcg ggttgggatc agcataagtt gtatctgggc agcttgcacggtttcaggca 360 ttttgttcat catttactca gatagtagtg ctgtcatcat ctgcctcatcaccatgttct 420 tcaccatgct ggctctcatg gcttctctct atgtccacat gttcctgatggccaggcttc 480 acattaagag gattgctgtc ctccccggca ctggtgccat ccgccaaggtgccaatatga 540 agggagcgat taccttgacc atcctgattg gcgtctttgt tgtctgctgggccccattct 600 tcctccactt aatattctac atctcttgtc ctcagaatcc atattgtgtgtgcttcatgt 660 ctcactttaa cttgtatctc atactgatca tgtgtaattc aatcatcgatcctctgattt 720 atgcactccg gagtcaagaa ctgaggaaaa ccttcaaaga gatcatctgttgctatcccc 780 tgggaggcct ttgtgacttg tctagcagat attaaatggg gacagagcacgcaatatagg 840 3 311 PRT Homo sapiens misc_feature (298)..(298) “Xaa”can be any amino acid 3 Gln Leu Phe Val Ser Pro Glu Val Phe Val Thr LeuGly Val Ile Ser 1 5 10 15 Leu Leu Glu Asn Ile Leu Val Ile Val Ala IleAla Lys Asn Lys Asn 20 25 30 Leu His Ser Pro Met Tyr Phe Phe Ile Cys SerLeu Ala Val Ala Asp 35 40 45 Met Leu Val Ser Val Ser Asn Gly Ser Glu ThrIle Ile Ile Thr Leu 50 55 60 Leu Asn Ser Thr Asp Thr Asp Ala Gln Ser PheThr Val Asn Ile Asp 65 70 75 80 Asn Val Ile Asp Ser Val Ile Cys Ser SerLeu Leu Ala Ser Ile Cys 85 90 95 Ser Leu Leu Ser Ile Ala Val Asp Arg TyrPhe Thr Ile Phe Tyr Ala 100 105 110 Leu Gln Tyr His Asn Ile Met Thr ValLys Arg Val Gly Ile Ser Ile 115 120 125 Ser Cys Ile Trp Ala Ala Cys ThrVal Ser Gly Ile Leu Phe Ile Ile 130 135 140 Tyr Ser Asp Ser Ser Ala ValIle Ile Cys Leu Ile Thr Met Phe Phe 145 150 155 160 Thr Met Leu Ala LeuMet Ala Ser Leu Tyr Val His Met Phe Leu Met 165 170 175 Ala Arg Leu HisIle Lys Arg Ile Ala Val Leu Pro Gly Thr Gly Ala 180 185 190 Ile Arg GlnGly Ala Asn Met Lys Gly Ala Ile Thr Leu Thr Ile Leu 195 200 205 Ile GlyVal Phe Val Val Cys Trp Ala Pro Phe Phe Leu His Leu Ile 210 215 220 PheTyr Ile Ser Cys Pro Gln Asn Pro Tyr Cys Val Cys Phe Met Ser 225 230 235240 His Phe Asn Leu Tyr Leu Ile Leu Ile Met Cys Asn Ser Ile Ile Asp 245250 255 Pro Leu Ile Tyr Ala Leu Arg Ser Gln Glu Leu Arg Lys Thr Phe Lys260 265 270 Glu Ile Ile Cys Cys Tyr Pro Leu Gly Gly Leu Cys Asp Leu SerSer 275 280 285 Arg Tyr Ala Pro Pro Glu Asn Asp Ile Xaa Val Ile Cys AsnPhe Ile 290 295 300 Asp Glu Asn Thr Ile Ala Leu 305 310 4 248 PRT Homosapiens 4 Lys Asn Leu His Ser Pro Met Tyr Phe Phe Ile Cys Ser Leu AlaVal 1 5 10 15 Ala Asp Met Leu Val Ser Val Ser Asn Gly Ser Glu Thr IleVal Ile 20 25 30 Thr Leu Leu Asn Ser Thr Asp Thr Asp Ala Gln Ser Phe ThrVal Asn 35 40 45 Ile Asp Asn Val Ile Asp Ser Val Ile Cys Ser Ser Leu LeuAla Ser 50 55 60 Ile Cys Ser Leu Leu Ser Ile Ala Val Asp Arg Tyr Phe ThrIle Phe 65 70 75 80 Tyr Ala Leu Gln Tyr His Asn Ile Met Thr Val Lys ArgVal Gly Ile 85 90 95 Ile Ile Ser Cys Ile Trp Ala Val Cys Thr Val Ser GlyVal Leu Phe 100 105 110 Ile Ile Tyr Ser Asp Ser Ser Ala Val Ile Ile CysLeu Ile Thr Val 115 120 125 Phe Phe Thr Met Leu Ala Leu Met Ala Ser LeuTyr Val His Met Phe 130 135 140 Leu Met Ala Arg Leu His Ile Lys Arg IleAla Val Leu Pro Gly Thr 145 150 155 160 Gly Thr Ile Arg Gln Gly Ala AsnMet Lys Gly Ala Ile Thr Leu Thr 165 170 175 Ile Leu Ile Gly Val Phe ValVal Cys Trp Ala Pro Phe Phe Leu His 180 185 190 Leu Ile Phe Tyr Ile SerCys Pro Gln Asn Pro Tyr Cys Val Cys Phe 195 200 205 Met Ser His Phe AsnLeu Tyr Leu Ile Leu Ile Met Cys Asn Ser Ile 210 215 220 Ile Asn Pro LeuIle Tyr Ala Leu Arg Ser Gln Glu Leu Arg Lys Thr 225 230 235 240 Phe LysGlu Ile Ile Cys Cys Tyr 245 5 20 DNA Sus scrofa 5 tggcaatagc caagaacaag20 6 20 DNA Sus scrofa 6 caggggatag caacagatga 20 7 18 DNA Sus scrofa 7ttaagtggag gaagaagg 18 8 19 DNA Sus scrofa 8 cattatgaca gttaagcgg 19 920 DNA Sus scrofa 9 taccctgacc atcttgattg 20 10 22 DNA Sus scrofa 10atagcaacag atgatctctt tg 22 11 24 PRT Sus scrofa 11 Met Ser His Phe AsnLeu Tyr Leu Ile Leu Ile Met Cys Asn Ser Ile 1 5 10 15 Ile Asp Pro LeuIle Tyr Ala Leu 20 12 24 PRT Homo sapiens 12 Met Ser His Phe Asn Leu TyrLeu Ile Leu Ile Met Cys Asn Ser Ile 1 5 10 15 Ile Asp Pro Leu Ile TyrAla Leu 20 13 24 PRT Rattus norvegicus 13 Met Ser His Phe Asn Leu TyrLeu Ile Leu Ile Met Cys Asn Ala Val 1 5 10 15 Ile Asp Pro Leu Ile TyrAla Leu 20 14 23 PRT Homo sapiens 14 Met Ser His Phe Asn Met Tyr Leu IleLeu Ile Met Cys Asn Ser Val 1 5 10 15 Ile Asp Pro Leu Ile Tyr Ala 20 1523 PRT Mammalia sp. 15 Met Ser His Phe Asn Met Tyr Leu Ile Leu Ile MetCys Asn Ser Val 1 5 10 15 Ile Asp Pro Leu Ile Tyr Ala 20 16 24 PRT Homosapiens 16 Met Ser Leu Phe Gln Val Asn Gly Val Leu Ile Met Cys Asn AlaIle 1 5 10 15 Ile Asp Pro Phe Ile Tyr Ala Leu 20 17 22 PRT Bos taurus 17Ala His Phe Asn Thr Tyr Leu Val Leu Ile Met Cys Asn Ser Val Ile 1 5 1015 Asp Pro Leu Ile Tyr Ala 20 18 22 PRT Homo sapiens 18 Ala His Phe AsnThr Tyr Leu Val Leu Ile Met Cys Asn Ser Val Ile 1 5 10 15 Asp Pro LeuIle Tyr Ala 20 19 23 PRT Homo sapiens 19 Met Ser His Phe Asn Met Tyr LeuIle Leu Ile Met Cys Asn Ser Val 1 5 10 15 Met Asp Pro Leu Ile Tyr Ala 2020 22 PRT Homo sapiens 20 Ser Tyr Phe Asn Leu Phe Leu Ile Leu Ile IleCys Asn Ser Val Val 1 5 10 15 Asp Pro Leu Ile Tyr Ala 20 21 25 PRT Bostaurus 21 Leu Ala Tyr Glu Lys Phe Phe Leu Leu Leu Ala Glu Phe Asn SerAla 1 5 10 15 Met Asn Pro Ile Ile Tyr Ser Tyr Arg 20 25 22 19 PRT Homosapiens 22 Phe Leu Leu Leu Ala Glu Ala Asn Ser Leu Val Asn Ala Ala ValTyr 1 5 10 15 Ser Cys Arg 23 22 PRT Homo sapiens 23 Val Phe Ala Phe CysSer Met Leu Cys Leu Leu Asn Ser Thr Val Asn 1 5 10 15 Pro Leu Ile TyrAla Leu 20 24 21 PRT Homo sapiens 24 Phe Gln Phe Phe Phe Trp Ile Gly TyrCys Asn Ser Ser Leu Asn Pro 1 5 10 15 Val Ile Tyr Thr Ile 20 25 22 PRTRattus norvegicus 25 Phe Asp Phe Val Val Ile Leu Thr Tyr Ala Asn Ser CysAla Asn Pro 1 5 10 15 Ile Leu Tyr Ala Phe Leu 20 26 16 PRT Homo sapiens26 Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe Leu 1 510 15

1. A method of identifying variation in a genotype of an animal, whereinsaid variation is associated with a phenotypic difference in meatquality traits of said animal, wherein said variation is a polymorphismcharacterized by an asparagine codon at an amino acid position analogousto amino acid 298 of a human MC4R gene product, wherein said animal withan asparagine codon at position 298 is indicative of said animal morelikely to have favorable meat quality characteristics than an animalwith an aspartic acid codon at position 298, said method comprising:obtaining a nucleic acid sample from said animal; assaying for thepresence of a polymorphism in a MC4R gene of said sample wherein saidpolymorphism at the amino acid level is characterized as a change froman aspartic acid codon to an asparagine codon at an amino acid positioncorresponding to amino acid 298 of a human MC4R gene product, whereinsaid animal with an asparagine codon is indicative of said animal morelikely to have favorable meat quality characteristics such as pH,marbling, color and drip loss than an animal with an aspartic acid codonat position 298; and relating said polymorphism to said phenotype. 2-3.(Cancelled).
 4. The method of claim 1 wherein the animal is a pig. 5.The method of claim 1 wherein the step of assaying for the presence ofthe polymorphism is a method employing allele specific oligonucleotides.6. The method of claim 1 wherein said polymorphism is assayed by PCRamplification and restriction.
 7. The method of claim 1 wherein the stepof assaying for the presence of the polymorphism employs a techniqueselected from the group consisting of restriction fragment lengthpolymorphism (RFLP) analysis, heteroduplex analysis, single strandconformational polymorphism (SSCP) analysis, denaturing gradient gelelectrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE),and use of linked genetic markers.
 8. The method of claim 7 wherein astep of assaying identifying the polymorphism comprises RFLP analysis.9. The method of claim 1 further comprising the step of: amplifying aregion of the MC4R gene sequence or a region therein which contains saidpolymorphism.
 10. The method of claim 9 further comprising the step ofdigesting the amplified region with a restriction endonuclease Taq I.11-18. (Cancelled).
 19. The method of claim 9 wherein said polymorphismis at position 678 of the amplification product produced by primers SEQID NO:5 and SEQ ID NO:6.
 20. A method of identifying an animal whichpossess a desired genotype indicative of a significantly correlatedphenotypic trait, the method comprising: obtaining a nucleic acid samplefrom an animal, said sample comprising a seventh transmembrane region ofan MC4R gene, and identifying the presence or absence of a Taq I site inone allele of the MC4R gene, wherein the presence of a Tag I siteindicates that the animal possesses a desired genotype indicative of aphenotypic trait selected from the group consisting of pH, marbling,color and drip loss.
 21. The method of claim 20 further comprising thestep of selecting animals with the desired genotype for breeding. 22.The method of claim 20 further comprising: amplifying the nucleic acidsample with a forward primer and a reverse primer; digesting theamplified nucleic acid with a restriction enzyme such that nucleic acidfragments are generated; separating the nucleic acid fragments accordingto size such that a restriction fragment pattern is generated whereinthe polymorphism is identifiable Taq I digestion pattern of an amplifiedproduct characterized by fragments of 466, 225, and 76 bp when a guanineis present at base 678 of the MC4R gene and fragments 542 and 225 bpwhen an adenine is present at base 678 of the amplified product.
 23. Themethod of claim 22 wherein the restriction fragment pattern ischaracterized by fragments of 156 and 70 bp when allele 1 of the MC4Rgene is present and a fragment of 226 bp when allele 2 of the MC4R geneis present, when a restriction enzyme that acts at the same recognitionsite as Taq I is used.
 24. The method of claim 20 wherein the animal isa pig.
 25. A kit for evaluating a nucleic acid sample from an animalcomprising: a reagent in a container that identifies a polymorphism in aMC4R gene.
 26. The kit of claim 25 wherein the reagent is a primer thatamplifies the MC4R gene or a fragment thereof.
 27. The kit of claim 25further comprising: a DNA polymerase which cleaves the MC4R gene, aforward primer, and a reverse primer, wherein the primers are capable ofamplifying a region of the MC4R gene which contains a polymorphic site.28. A primer for assaying the presence of a polymorphic Taq I site inthe MC4R gene wherein the primer comprises a sequence selected from thegroup consisting of SEQ. ID NO:6, SEQ. ID NO:7, SEQ. ID NO:8, SEQ. IDNO:9, SEQ. ID NO:10, and SEQ. ID NO:11.
 29. A method for selectinganimals for the desired traits of favorable meat quality comprising thesteps of: obtaining a nucleic acid sample from an animal, identifying apolymorphism said polymorphism being a nucleotide substitution atposition 678 of SEQ ID NO: 1, and selecting the animals which have thenucleotide associated with a desired trait selected from the groupconsisting of pH, marbling, color and drip loss at position 678, whereinthe presence of the polymorphism indicates that the animal possesses agenotype indicative of a favorable meat quality selected from the groupconsisting of pH, marbling, color, and drip loss.
 30. A method forindirect selection for a polymorphism in an MC4R gene comprising:obtaining a nucleic acid sample from an animal, and identifying anucleotide substitution at position 678 of SEQ ID NO: 1 with a DNAmarker known to be associated with MC4R gene, said DNA marker furtherbeing associated with meat quality, wherein specific alleles of the DNAmarker are used to make the indirect identification of the nucleotidesubstitution, and selecting said animals based upon the presence ofnucleotide substitution.
 31. The method of claim 30 wherein the linkedmarker is selected from the group consisting of S033 1, BHT0433, andS0313.
 32. A method of identifying animals which possess a desiredgenotype indicative of favorable meat quality traits, the methodcomprising: determining an association between a MC4R genotype and atrait of interest by obtaining a sample of animals from a line or breedof interest, preparing a nucleic acid sample from each animal in thesample, determining the genotype of the MC4R gene by screening for apolymorphism being a point mutation in the 7^(th) transmembrane domainof the MC4R gene, wherein the presence of the polymorphism indicatesthat the animal possesses a genotype indicative of favorable meatquality characteristics selected from the group consisting of pH,marbling, color, and drip loss and calculating the association betweenthe MC4R genotype and the trait.
 33. A method of selecting animals whichpossess a desired MC4R genotype indicative of a significantly associatedphenotypic trait, the method comprising: obtaining a nucleic acid samplefrom an animal by screening for a polymorphism being a G to A pointmutation at position 678 of SEQ ID NO:1 from the MC4R gene, identifyingthe genotype of the MC4R gene of the animal, and selecting those animalswhich have the genotype associated with the desired traits, wherein thepresence of the polymorphism indicates that the animal possesses agenotype previously shown to be significantly associated with saiddesired trait.
 34. A method of determining the potential meat quality ofan animal, said method comprising: obtaining a nucleic acid sample fromsaid animal and then assaying for the presence of a polymorphism in theMC4R gene of the sample, said polymorphism being a G to A point mutationat position 678 of SEQ ID NO: 1 from the MC4R gene, said polymorphismbeing one which is associated with favorable meat qualitycharacteristics selected from the group consisting of pH, marbling,color, and drip loss, wherein the presence of the polymorphism indicatesthat the animal possesses a genotype indicative of favorable meatquality.
 35. A method of selecting animals for breeding, said methodcomprising: obtaining a nucleic acid sample from said animal; assayingfor the presence of a polymorphism in the MC4R gene of said sample, saidpolymorphism being a G to A point mutation at position 678 of SEQ IDNO:1 from the MC4R gene, said polymorphism being correlated with aphenotypic trait; and using the MC4R genotype as part of a selectionmodel based on the estimated value of the effect of the marker genotype,and thereafter selecting animals on the basis of this estimated valuefor use in breeding.
 36. A method of segregating animals in order toprovide uniformity at slaughter comprising: obtaining a nucleic acidsample from said animal; and assaying for the presence of a polymorphismin the MC4R gene of said sample, said polymorphism being a G to A pointmutation at position 678 of SEQ ID NO:1 from the MC4R gene, saidpolymorphism being one which is associated with meat quality,segregating said animals based upon the polymorphism present in saidanimal.
 37. A method for selecting animals for breeding, said methodcomprising: obtaining a nucleic acid sample from said animal; assayingfor the presence of a polymorphism in the seventh transmembrane domainof the MC4R gene of said sample said polymorphism being one which isassociated with a phenotypic trait; and selecting animals with afavorable allele for inclusion in breeding stock.
 38. (Cancelled) 39.(Cancelled)
 40. A method for selecting pigs for the desired trait ofimproved meat quality comprising the steps of: obtaining a nucleic acidsample from a pig, screening said pig for a polymorphism comprising a Gto A mutation at position analogous to position 678 of SEQ ID NO: 1 fromthe MC4R gene, said polymorphism being one which is associated with afavorable meat quality characteristic, and selecting said pig which havethe nucleotide associated with the desired traits at position 678.